Plants and plant-derived products have long been popular complementary health interventions in America and throughout the world. Recently, there is increased evidence that plant RNAs ingested in the course of eating ordinary plant-based foods are taken up by the mammalian digestive tract and remain functional. These observations raise the possibility of incorporating delivery of modern molecular RNA-based therapeutics into what has traditionally been a complementary health intervention modality. MicroRNAs (miRNAs) are a class of small non-coding RNAs that play a critical role in gene expression in eukaryotes, controlling virtually every physiological process in the body. Reduction in certain miRNAs is associated with many diseases, and restoration of the missing miRNA blocks disease progression. Although the therapeutic potential of these miRNAs is clear, delivery of the needed miRNA to the diseased cells is a critical barrier to implementation of the therapy. This R21 proposal is focused on developing a safe, effective, and economically feasible strategy for delivering therapeutic miRNAs in vivo. The strategy is based on the scientific premise that edible plants can be used as biofactories to produce therapeutic mammalian miRNAs that can be administered via ingestion. This strategy represents a fundamental shift from the current mainstream approach in the field, which has focused on synthesizing miRNAs as double-stranded ?miRNA mimics? and delivering them intravenously, formulated with lipids or liposomes. The idea of using plants to produce therapeutic miRNAs is particularly attractive for several reasons. Plants can be bioengineered to make miRNAs of any desired sequence and plant-made miRNAs are biologically natural - not double-stranded or chemically modified. Furthermore, plants package miRNAs into nanoparticles that resemble exosomes. These plant exosomes stabilize miRNAs in the GI tract and are taken up via intestinal stem cells and macrophages, providing an effective delivery mechanism. Finally, the plant exosomal membranes themselves have been shown to have anti- inflammatory properties. The proposed experiments build on our preliminary results from feeding experiments using plant-produced mammalian tumor suppressor miRNAs to reduce tumor burden in the well-established Apcmin/+ mouse model of colon cancer. The goals of the proposal are three-fold: 1) Characterize the miRNA content of exosomes isolated from our transgenic plants that have been bioengineered to produce validated mammalian tumor suppressor miRNAs; 2) establish methods to enhance packaging of bioengineered mammalian miRNAs into the plant exosomal fraction; and 3) test the efficacy of orally administered tumor suppressor miRNA-containing plant exosomes in chemoprevention, using the APCmin/+ mouse model of colon cancer. To our knowledge, we are the only group exploring this potentially transformative approach to enabling the use of miRNA therapeutics.